Amplification of nucleic acid molecules is routinely practiced in medical and bioresearch settings for a variety of tasks, such as the detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of infectious diseases, the cloning of genes, paternity testing, and other types of nucleic acid analysis. Nucleic acid amplification techniques include, but are not limited to, PCR, the ligase chain reaction (LCR), the transcription based amplification system (TAS), the nucleic acid sequence-based amplification (NASBA), the strand displacement amplification (SDA), rolling circle amplification (RCA), and hyper-branched RCA (HRCA).
Digital amplification is a technique that allows quantitative measurement of the number of target molecules in a sample. The basic premise of the technique is to divide a large sample into a number of smaller subvolumes (partitioned volumes), whereby the subvolumes contain on average a low number or single copy of target. Then, by counting the number of successful amplification reactions in the subvolumes, one can deduce the starting copy number of the target in the sample.
In some cases, target nucleic acids can be difficult to amplify. Amplification reactions that fail for reasons unrelated to the presence or absence of target nucleic acid in a bulk solution will distort statistical analysis of the results, and result in an incorrect determination of the quantity of target molecules present in a sample. For example, RNA molecules which are typically subject to a reverse transcription reaction prior to amplification can confound quantitative analysis by amplification due to bias introduced during the reverse transcription step. Sources of bias introduced by reverse transcription can include poor processivity and fidelity of the reverse transcriptase, RNA secondary structure, and degradation or poor quality of target RNA molecules. RNAs that are readily transcribed into DNA can be artificially increased in apparent abundance, while RNAs that are difficult to transcribe into DNA can be artificially reduced in apparent abundance. Generally multiple rounds of reverse transcription exacerbate bias because RNA molecules that fail to be transcribed into DNA during one round are likely to fail during subsequent rounds of reverse transcription. Additionally, RNA molecules that are transcribed readily during one round, are likely to be transcribed readily during subsequent rounds. Thus, multiple rounds of reverse transcription in a bulk solution can further artificially increase the apparent abundance of readily transcribed RNAs and artificially decrease the apparent abundance of RNAs that are difficult to transcribe.